Lipopolyamines, their preparation and their use

ABSTRACT

New lipopolyamines of general formula (I), their salts, their preparation and their use. ##STR1## n=1 to 5 and m=2 to 6; R represents a radical ##STR2## (R 1  and R 2  : aliphatic radical containing 12 to 22 carbon atoms; R: hydrogen atom or alkyl radical optionally substituted with phenyl), or a radical ##STR3## (X=CH 2 , CO; R 3  and R 4  aliphatic radical containing 11 to 21 carbon atoms), their preparation and their use. 
     The lipopolyamines of general formula (I) are especially useful as vectors for the transfection of eukaryotic cells.

The present invention relates to new lipopolyamines of general formula:##STR4## in the D, L or DL form, to their salts, to their preparationand to their use.

In the general formula (I),

n is an integer between 1 and 5 inclusive,

m is an integer between 2 and 6 inclusive,

R represents a hydrogen atom or a radical of general formula: ##STR5##in which R₁ and R₂, which may be identical or different, each representa saturated aliphatic radical C_(p) H_(2p+2) or unsaturated aliphaticradical C_(p) H_(2p) or C_(p) H_(2p-2), p being an integer between 12and 22 inclusive, and R' represents a hydrogen atom or an alkyl radicalcontaining 1 to 4 carbon atoms optionally substituted with a phenylradical, or a radical of general formula: ##STR6## in which X representsa methylene group (--CH₂ --) or a carbonyl group (--CO--), and R₃ andR₄, which may be identical or different, each represent a saturatedaliphatic radical C_(p') H_(2p'+2) or unsaturated aliphatic radicalC_(p') H_(2p') or C_(p') H_(2p'-2), p' being an integer between 11 and21 inclusive, on the understanding that:

irrespective of the values of m and n, only one of the symbols Rrepresents a radical of general formula (II) or (III)

when n is between 2 and 5, the values of m in the different fragments##STR7## may be identical or different.

Of very special interest are the products of general formula (I) inwhich n is equal to 3 and the values of m in the fragments ##STR8## areidentical or different and represent 3 or 4, and R represents: either aradical of general formula (II) in which R₁ and R₂ each represent analkyl radical containing 12 to 22 carbon atoms and R' represents ahydrogen atom, or a radical of general formula (III) in which R₃ --X--and R₄ --X-- each represent an alkanoyl radical containing 12 to 22carbon atoms.

Of still more special interest are5-carboxyspermylglycinedioctadecylamide (DOGS) anddipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).

According to the invention, the new lipopolyamines of general formula(I) may be obtained by the reaction of a product of general formula:##STR9## in which m and n are defined as above, R₅ represents a hydrogenatom or a carboxyl radical and the symbols Z represent a groupprotecting the amine function, on the understanding that:

irrespective of the values of m and n, only one of the symbols R₅represents a carboxyl radical

when n is between 2 and 5, the values of m in the different fragments##STR10## may be identical or different either with a product of generalformula: ##STR11## in which R₁, R₂ and R' are defined as above, or witha product of general formula: ##STR12## in which R₃, R₄ and X aredefined as above, followed by replacement of the protective groups Z bya halogen atom.

When a product of general formula (V) is reacted with a product ofgeneral formula (IV), it is especially advantageous to perform thecondensation in the presence of a diimide such asdicyclohexylcarbodiimide, working in an inert organic solvent selectedfrom halogenated aliphatic solvents such as methylene chloride.

When a product of general formula (VI) is reacted with a product ofgeneral formula (IV), it is especially advantageous to treat the acidfunction of the product of general formula (IV) beforehand withN-hydroxysuccinimide, working in an organic solvent selected fromhalogenated aliphatic hydrocarbons (methylene chloride) and ethers(tetrahydrofuran) in the presence of an imide such as dicyclohexylcarbodiimide, before performing the condensation of the product ofgeneral formula (VI). The condensation of the mixed ester with theproduct of general formula (VI) is generally performed in an organicsolvent (chloroform, ethanol) in the presence of an organic base such astriethylamine at a temperature of between 30° and 50° C.

A protective group Z which is readily replaceable by a hydrogen atomwithout affecting the remainder of the molecule is generally used. It isespecially advantageous to use as a protective group thet-butoxycarbonyl radical, which is readily replaceable by a hydrogenatom by means of an acid (trifluoroacetic acid).

The products of general formula (IV) in which n is greater than 1 may beobtained from ornithine by cyanoalkylation followed by reduction of thenitrile functions to amine functions and then protection of the aminefunctions thereby obtained.

The products of general formula (V) may be obtained by the reaction ofan amine of general formula: ##STR13## in which R₁ and R₂ are defined asabove, with an amino acid of general formula: ##STR14## in which R' isdefined as above, in which the amine function is protected and in whichthe acid function is activated. It is especially advantageous to protectthe amine function by a benzyloxycarbonyl radical, which is readilyreplaceable by a hydrogen atom by hydrogenolysis in the presence of acatalyst such as palladium. The acid function is generally activated byconversion to a p-nitrophenyl ester.

The products of general formula (VI) are known products which arereadily accessible.

The new products of general formula (I) have the property, whendispersed in water, of forming unilamellar nanoparticles which areunstable in an ionic medium and which associate strongly, via theircationic portion, with plasmid or oligonucleotide DNA, compacting thelatter and covering it with a lipid layer. By using an excess ofcationic charges relative to the nucleic acid, the lipid/DNA complexesmay be adsorbed on cell membranes, therby facilitating uptake of the DNAby the cells.

The products of general formula (I) constitute specific, non-toxic,biodegradable vectors of great efficacy for carrying out thetransfection of eukaryotic cells (cell lines, primary cultures).

According to the invention, transfection is carried out by bringing acell suspension in the absence of serum into contact with a transfectingmixture obtained, at the time of use, from a solution of thelipopolyamine of general formula (I) and a solution of the DNA in asuitable medium.

It is especially advantageous to work in a very dilute medium (1 to 5nanomolar) and to use an excess (from 2- to 5-fold) of charges of thelipopolyamine relative to the DNA.

The transfection time can be between 10 minutes and 48 hours,independently of the nature of the cells.

The method according to the invention has the advantage of beingapplicable to cell lines of diverse origins (including, for example,LMKT, Ras4, CHO, F9, Bu4, S49, Hela and AtT20) as well as to primarycells, without the need to optimize or modify the conditions forcarrying out the method.

Moreover, the lipopolyamines of general formula (I) enable fragile cells(intermediate or anterior hypophyseal cells, chromaffin cells,peripheral or central neurons), which it was not possible to transfectby the application of classical methods (calcium phosphatecoprecipitation or dextran techniques), to be transfected.

Finally, the transfection agents according to the invention do notmanifest toxicity with respect to the transfected cells. They do notmanifest acute toxicity in rats after intracerebral or systemicinjection.

The subject of the present invention is also a stable alcoholic oraqueous solution of a lipopolyamine of general formula (I) which isusable for carrying out cell transfections.

Solutions containing 1 mg/ml, which enable approximately 50transfections to be carried out, are generally prepared.

The examples which follow, given without implied limitation, show howthe invention may be put into practice.

EXAMPLE 1

A mixture of L-5-carboxytetra(t-butoxycarbonyl)spermine (1 equivalent)and glycinedioctadecylamide (1 equivalent) in methylene chloride isstirred for 12 hours in the presence of dicyclohexylcarbodiimide (1.1equivalent).

After chromatography on silica,tetra(t-butoxycarbonyl)-5-carboxyspermylglycinedioctadecylamide isobtained in a 90% yield, the protective groups of which product arremoved by treatment with trifluoroacetic acid for 10 minutes at atemperature in the region of 20° C. The tetrakis(trifluoroacetate) of5-carboxyspermylglycinedioctadecylamide (DOGS) is thereby obtained.

The structure of the product obtained is confirmed by the proton nuclearmagnetic resonance spectrum at 200 MHz in deuterated methanol (chemicalshifts δ in ppm): 0.9 [t, (CH₃)₂ ]; 1.3 [m, 2×(CH₂)₁₅ ]; 1.4-1.7 (m,2×CH₂ CH₂ NCO); 1.8-2.2 (m, 4×CH₂ CH₂ N⁺); 3.0-3.2 (m, 5×CH₂ N⁺); 3.35(t, 2×CH₂ NCO); 4.0 (t, CHN⁺); 4.15 (s, COCH₂ ND).

L-5-Carboxytetra(t-butoxycarbonyl)spermine may be prepared in thefollowing manner:

2.2 equivalents of acrylonitrile are added to a 1M solution ofL-ornithine in dimethylformamide. The mixture is stirred for 1 hour at atemperature in the region of 20° C.

The dinitrile thereby obtained is reduced with hydrogen in the presenceof Raney nickel, working in the presence of ethanolic potassiumhydroxide, to give L-5-carboxyspermine, the amine functions of which areprotected by t-butoxycarbonyl groups by application of the usualmethods.

Glycinedioctadecylamide may be prepared by the condensation ofdioctadecylamine (1 equivalent) with N-carbobenzoxyglycine p-nitrophenylester (1 equivalent), working in methylene chloride in the presence oftriethylamine (1.1 equivalent) for 5 hours.

After hydrogenation for 1 hour in the presence of palladium on charcoal(10% palladium), working in a methylene chloride/ethanol mixture,glycinedioctadecylamide is obtained in an 87% yield.

EXAMPLE 2

L-5-Carboxytetra(t-butoxycarbonyl)spermine (1 equivalent) is treated for12 hours with N-hydroxysuccinimide (1.1 equivalent) in the presence ofdicyclohexylcarbodiimide (1.1 equivalent), working in a methylenechloride/tetrahydrofuran mixture.

The ester obtained is treated with dipalmitoylphosphatidylethanolamine(1 equivalent) in the presence of triethylamine (1 equivalent) in achloroform/ethanol mixture for 12 hours at 40° C. After treatment of thereaction mixture, dipalmitoylphosphatidylethanolaminetetra(t-butoxycarbonyl)-5-carboxyspermylamide is obtained in a 55%yield, the protective groups of which product are removed withtrifluoroacetic acid in methylene chloride.Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES) isthereby obtained in the form of a tetrakis(trifluoroacetate).

The structure of the product obtained is confirmed by the proton nuclearmagnetic resonance spectrum at 200 MHz in a deuteratedchloroform/deuterated methanol mixture (1:1 by volume) (chemical shiftsδ in ppm): 0.85 [t, (CH₃)₂ ]; [m, 2×(CH₂)₁₂ ]; 1.5-1.65 (m, 2×CH₂ CO₂);1.8-2.1 (m, 4×CH₂ CH₂ N⁺); 2.3 (tt, 2×CH₂ CH₂ CO₂); 2.9-3.1 (m, 5×CH₂N⁺); 3.2 (bm, CH₂ NDCO); 3.75-4.05 (m, CHN⁺, 2×CH₂ OP); 4.15-4.40 (2×dd,CO₂ CH₂); 5.20 (OCH).

EXAMPLE 3

A 20 mM solution of DOGS in ethanol is diluted 10-fold with sterilewater so as to obtain a 2 mM solution. 7.5 μl of this solution arewithdrawn and diluted in 250 μl of DMEM medium (Dulbecco ModifiedEssential Medium).

A solution is prepared containing 5 μg of plasmid containing a vectorfor the expression of chloramphenicol acetyltransferase (CAT) (forexample a construction derived from plasmid pCAT 8+ [L. Klein-Hitpass etal., Cell, 46, 1053-1061 (1986)] by the insertion of a fragment(BamHI-XbaI) containing 4 copies of the AP1 sequence ["binding consensussequence"] (pCAT 4XB)]) in 250 μl of DMEM medium.

A suspension is prepared of 10⁵ -10⁶ melanotropic cells [preparedaccording to B. A. Demeineix et al., Neuroscience, 17, 1275-1285 (1986)]in 500 μl of DMEM medium in the absence of serum.

The DOGS and plasmid solutions are mixed and the "transfecting" mixtureis added to the cell suspension.

The mixture is incubated at 37° C. for a specified period.

After transfection, the cells are washed twice and then plated. After 48hours, the cells are washed with phosphate buffer (PBS) and thechloramphenicol acetyltransferase activity is then determined accordingto the method of C. M. Gorman et al., Mol. Cell. Biol., 2, 1044-1051(1982).

The cells are resuspended in 100 μl of a 200 mM solution of Tris-HCl (pH7.4). After several cooling/heating cycles, 50 μl of the supernatant areadded to 40 μl of Tris-HCl (pH 7.4) containing ¹⁴ C-labelledchloramphenicol (0.1 μCi). After 5 minutes at 37° C., the reaction isinitiated by adding 20 μl of acetyl-COA (4 mM). After 1 hour at 37° C.,the chloramphenicol and its acetylated derivatives are extracted withethyl acetate, separated by thin-layer chromatography andautoradiographed. The autoradiograms are analysed by a suitable method.

This method thus enables different promoters to be analysed in primarycultures in general.

We claim:
 1. A method of transfecting eukaryotic cells, comprisingmixing a dilute aqueous solution of a lipopolyamine product selectedfrom the group consisting of 5-Carboxyspermylglycinedioctadecylamide andDipalmitoylphosphatidylethanolamine 5-carboxyspermylamide in a suitablemedium with a dilute aqueous solution of plasmid or oligonucleotide DNAto form a transfecting solution and contacting the transfecting solutionwith a suspension of cells to be treated.
 2. The method of claim 1,wherein cell lines or primary cultures are transfected. 3.5-Carboxyspermylglycinedioctadecylamide. 4.Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide.
 5. Anethanolic solution for carrying out the transfection of eukaryotic cellscomprising mixing a dilute aqueous solution of a product selected fromthe group consisting of 5-Carboxyspermylglycinedioctadecylamide andDipalmitoylphosphatidylethanolamine 5-carboxyspermylamide in a suitablemedium with a dilute aqueous solution of plasmid or oligonucleotide DNA,and the transfecting mixture is then brought into contact with asuspension of cells to be transfected.
 6. An ethanolic solution forcarrying out the transfection of eukaryotic cells comprising mixing adilute aqueous solution of a product selected from the group consistingof 5-Carboxyspermylglycinedioctadecylamide andDipalmitoylphosphatidylethanolamine 5-carboxyspermylamide in a suitablemedium with a dilute aqueous solution of a plasmid or oligonucleotideDNA, and the transfecting mixture is then brought into contact with asuspension of cell lines or primary cultures to be transfected.
 7. Anaqueous solution for carrying out the transfection of eukaryotic cellscomprising mixing a dilute aqueous solution of a product selected fromthe group consisting of 5-Carboxyspermylglycinedioctadecylamide andDipalmitoylphosphatidylethanolamine 5-carboxyspermylamide in a suitablemedium with a dilute aqueous solution of plasmid or oligonucleotide DNA,and the transfecting mixture is then brought into contact with asuspension of cells to be transfected.
 8. An aqueous solution forcarrying out the transfection of eukaryotic cells comprising mixing adilute aqueous solution of a product selected from the group consistingof 5-Carboxyspermylglycinedioctadecylamide andDipalmitoylphosphatidylethanolamine 5-carboxyspermylamide in a suitablemedium with a dilute aqueous solution of plasmid or oligonucleotide DNA,and the transfecting mixture is then brought into contact with asuspension of cell lines or primary cultures to be transfected.